The cytoskeletal and/or nuclear matrix molecules responsible for morphological changes associated with apoptosis were identified using monoclonal antibodies (mAbs). We developed mAbs against Triton X-100-insoluble components of HL-60 cells pretreated with all-trans retinoic acid. In particular, one mAb recognized a 22-kDa protein that exhibited intriguing behavior by forming an aggregate and appearing as a speck during apoptosis induced by retinoic acid and other anti-tumor drugs. Cloning and sequencing of its cDNA revealed that this protein comprises 195 amino acids and that its Cterminal half has a caspase recruitment domain (CARD) motif, characteristic of numerous proteins involved in apoptotic signaling. We referred to this protein as ASC (apoptosis-associated speck-like protein containing a CARD). The ASC gene was mapped on chromosome 16p11.2-12. The antisense oligonucleotides of ASC were found to reduce the expression of ASC, and consequently, etoposide-mediated apoptosis of HL-60 cells was suppressed. Our results indicate that ASC is a novel member of the CARD-containing adaptor protein family.

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The pyrin domain was identified recently in multiple proteins that are associated with apoptosis and/or inflammation, but the physiological and molecular function of these proteins remain poorly understood. We have identified Caspy and Caspy2, two zebrafish caspases containing N-terminal pyrin domains. Expression of Caspy and Caspy2 induced apoptosis in mammalian cells that were inhibited by general caspase inhibitors. Biochemical analysis revealed that both Caspy and Caspy2 are active caspases, but they exhibit different substrate specificity. Caspy, but not Caspy2, interacted with the zebrafish orthologue of ASC (zAsc), a pyrin-and caspase recruitment domain-containing protein identified previously in mammals. The pyrin domains of both Caspy and zAsc were required for their interaction. Furthermore, zAsc and Caspy co-localized to the "speck" when co-transfected into mammalian cells. Enforced oligomerization of zAsc, but not simple interaction with zAsc, induced specific proteolytic activation of Caspy and enhanced Caspy-dependent apoptosis. Injection of zebrafish embryos with a morpholino antisense oligonucleotide corresponding to caspy resulted in an "open mouth" phenotype associated with defective formation of the cartilaginous pharyngeal skeleton. These studies suggest that zAsc mediates the activation of Caspy, a caspase that plays an important role in the morphogenesis of the jaw and gill-bearing arches.

Of 11 children with juvenile myelomonocytic leukemia (JMML) carrying RAS mutations (8 with NRAS mutations, 3 with KRAS2 mutations), 5 had a profound elevation in either or both the white blood cells and spleen size at diagnosis. Three patients had no or modest hepatosplenomegaly and mild leukocytosis at presentation but subsequently showed a marked increase in spleen size with or without hematologic exacerbation, for which nonintensive chemotherapy was initiated. The other three patients with NRAS or KRAS2 glycine to serine substitution received no chemotherapy, but hematologic improvement has been observed during a 2-to 4-year follow up. In the third group, all hematopoietic cell lineages analyzed had the RAS mutations at the time of hematologic improvement, whereas DNA ob- IntroductionSomatic point mutations of the RAS genes at codons 12, 13, and 61 (NRAS and KRAS2) are found in approximately 20% of patients with juvenile myelomonocytic leukemia (JMML). 1,2 Other patients show inactivation of NF1 or PTPN11 mutations. [3][4][5] Although most patients with JMML die from progressive disease unless treated with hematopoietic stem cell transplantation, there are a few patients who have been reported to spontaneously recover without intervention. 6,7 Some of these children have JMML associated with Noonan syndrome, but others do not. So far, the individual prognosis in JMML-carrying specific genetic aberrations remains unclear. We report the clinical course in 11 patients with RAS mutations. Materials and methodsThis study was approved by the Institutional Review Board of Shinshu University. Informed consent was obtained from the guardians of the patients following institutional guidelines. Cell preparationWe used peripheral blood (PB) or bone marrow (BM) mononuclear cells (MNCs) that had been frozen with liquid nitrogen. CD3-and CD56-positive PB cells were separated immunomagnetically. 8 Ninety-nine percent of the isolated cells were CD3-or CD56-positive according to a flow cytometric analysis. Clonal cell cultureTwenty thousand PB or BM MNCs were plated in a dish containing methylcellulose medium supplemented with or without 0.01 to 10 ng/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF). 9 To examine the clonal derivation of myeloid and erythroid lineages, 2000 CD34 ϩ PB cells harvested immunomagnetically were cultured in methylcellulose medium supplemented with GM-CSF, stem cell factor, interleukin 3, and erythropoietin. Twelve days after incubation in 5% CO 2 , GM colonies, erythroid colonies, and mixed colonies were individually lifted and prepared as single cell suspensions. Sequence analyses were then performed on individual colonyconstituent cells. Detection of NRAS and KRAS2 mutationsDNA was extracted from PB or BM MNCs and nails. Exon 1 (codons 12 and 13) and exon 2 (codon 61) of NRAS and KRAS2 genes were amplified by polymerase chain reaction (PCR) using primer pairs described previously. 10,11 The PCR products were subjected to direct sequencing from both directions on an automatic DNA se...

SummaryOrganisms of the Mycobacterium avium complex (MAC) are widely distributed in the environment, form biofilms in water pipes and potable water tanks, and cause chronic lung infections in patients with chronic obstructive pulmonary disease and cystic fibrosis. Pathological studies in patients with pulmonary MAC infection revealed granulomatous inflammation around bronchi and bronchioles. BEAS-2B human bronchial epithelial cell line was used to study MAC invasion. MAC strain A5 entered polarized BEAS-2B cells with an efficiency of 0.1 ± ± ± ± 0.03% in 2 h and 11.3 ± ± ± ± 4.0% in 24 h. In contrast, biofilm-deficient transposon mutants 5G4, 6H9 and 9B5 showed impaired invasion. Bacteria exposed to BEAS-2B cells for 24 h had greater ability to invade BEAS-2B cells compared with bacteria incubated in broth. M. avium had no impact on the monolayer transmembrane resistance. Scanning electron microscopy showed that MAC A5 forms aggregates on the surface of BEAS-2B cell monolayers, and transmission electron microscopy evidenced MAC within vacuoles in BEAS-2B cells. Cells infected with the 5G4 mutant, however, showed significantly fewer bacteria and no aggregates on the cell surface. Mutants had impaired ability to cause infection in mice, as well. The ability to form biofilm appeared to be associated with the invasiveness of MAC A5.